Full-Length RNA Sequencing Frequently Asked Questions

Full-length RNA sequencing captures full-length transcripts (often exceeding 10 kb) using long-read sequencing technologies such as PacBio® and Oxford Nanopore Technologies® sequencing platforms.

PacBio® Kinnex full-length RNA sequencing uses cDNA-based sequencing with high-accuracy HiFi reads (Q20–Q30+).

Oxford Nanopore Technologies® direct RNA sequencing uses native RNA that preserves modifications.

This service is ideal for:

• Isoform discovery and quantification
• Alternative splicing and exon usage
• Fusion gene detection (especially in cancer)
• Allele-specific expression
• Sequence integrity for RNA therapeutics (download our Tech Note to learn more)
• Transcriptome annotation in non-model organisms
• Single-cell isoform sequencing (PacBio® Kinnex single-cell RNA-Seq, Oxford Nanopore Technologies® cDNA-PCR)

Please submit a quote request to receive accurate pricing information, as the cost depends on the details of the project.

You can also contact the NGS team by submitting an inquiry.

We offer full-length RNA sequencing on PacBio® and Oxford Nanopore Technologies® sequencing platforms. Platform selection depends on project needs including accuracy and throughput.

Unsure which platform to choose? Please submit an inquiry for assistance with designing your experiment.

PacBio® HiFi reads offer superior per-base accuracy (>99.9%) due to circular consensus sequencing, while Oxford Nanopore Technologies® has improved significantly with newer basecallers but still trails slightly in raw accuracy. GENEWIZ provides Super Accuracy Basecalling (SUP) for Oxford Nanopore Technologies®, achieving high read accuracy often exceeding 97%, and enhancing the detection of RNA modifications.

Yes, both platforms support barcoding for multiplexing. PacBio® uses barcoded hairpin adapters, while Oxford Nanopore Technologies® uses rapid and native barcoding kits.

Data output will vary depending on sequencing platform. PacBio® typically yields 3–4 million HiFi reads per SMRT Cell (Revio), with full-length transcript coverage. Oxford Nanopore Technologies® output varies by flow cell (PromethION, GridION), ranging from 10–100 million reads depending on run time and library complexity.

We can accept high-quality total RNA. Additionally, GENEWIZ can perform extractions from a wide range of starting materials, including cell pellets, fresh frozen tissue, and other sample types.

For detailed sample submission requirements, please visit our sample submission guidelines.

Oxford Nanopore Technologies® is preferred for detecting native RNA modifications (e.g., m6A) since it sequences RNA directly without reverse transcription.

Yes. PacBio® offers Kinnex single-cell RNA for 10x Genomics® libraries, and Oxford Nanopore Technologies® supports single-cell cDNA sequencing with custom workflows. Please submit an inquiry for assistance with designing your experiment.

The GENEWIZ NGS team is composed of Ph.D. scientists who can help you optimize your project design and provide consultation. You can contact the team by submitting an inquiry.

Our team prioritizes fast turnaround times and provides a timeline based on first-pass processing. The turnaround time listed within the quote is inclusive of all steps quoted, unless otherwise noted. If repeat processing is required, the turnaround may be subject to change and will be proactively communicated to the client.

If the scope of a project changes after project initiation or if sample or project clarification is required after sample receipt, GENEWIZ may reassess the turnaround time based on the subsequent communications and modifications (if applicable).

All extraction kits and reagents are routinely updated to remain best-in-class. If you are seeking access to previous versions of our method, please submit an inquiry (availability is assessed on a case-by-case basis).

Extractions are performed to the best of our ability, and we are unable to guarantee yield due to multiple variables that may affect sample yield and quality. Costs to cover the work performed will be applied, regardless of the outcome.

All preparation kits and reagents are routinely updated to remain best-in-class. If you are seeking access to previous versions of our method, please submit an inquiry (availability is assessed on a case-by-case basis).

For most of the services, an initial sample QC is included within the fee of the project for all samples that will proceed to the next processing stage (e.g. library preparation or sequencing) unless otherwise noted within the quotation. Resubmissions or additional, optional samples may incur a nominal fee for QC.

Initial sample QC can include:

• Assessing RNA concentration and integrity
• Assessing DNA concentration and DNA size (for select projects)
• Assessing premade library size and concentration
• Assessing cryopreserved cells by cell count and viability

The sequencing platform will be listed within the Service Description of the quotation.

Unless specifically noted in the quotation, GENEWIZ reserves the right to choose between equivalent instruments depending on the target read depth and configuration requested. If a specific instrument is required, please add special comments in your quote/order and notify the GENEWIZ NGS team prior to project initiation.

You can contact the GENEWIZ NGS team at:

• US: NGS@azenta.com
• EU & UK: NGS.Europe@azenta.com

Illumina-based projects

For samples that pass QC and libraries prepared at GENEWIZ:

Data Quality

• NovaSeq 2x150bp: ≥85% of bases ≥Q30
• NovaSeq 2x250bp: ≥80% of bases ≥Q30
• MiSeq 2x150bp: ≥80% of bases ≥Q30
• MiSeq 2x250bp: ≥75% of bases ≥Q30

Data Yield

• Within 10% of total data target yield per lane or flowcell, unless otherwise noted
• Within 20% of per sample target yield, unless otherwise noted

Quality and yield for samples that do not pass QC and are processed at best effort are not guaranteed. Premade libraries/library pools submitted for sequencing only quality and yield are evaluated on a case-by-case basis.

Multiplexing is performed to the best of our ability to ensure relatively even data distribution amongst samples.

PacBio-based projects

Due to various sample-related factors that may influence long-read sequencing yield and quality, GENEWIZ cannot guarantee overall data output and quality. However, based on extensive experience with long-read workflows, GENEWIZ has established target metrics based on the PacBio system based on the sample submitted and library type. If a project does not meet these targets, a thorough review of the processing will be performed. If the issue is determined to be unrelated to the sample, a repeat or top-off will be performed as necessary.

Due to the possible wide range of performance being influenced by sequence complexity and sample quality, GENEWIZ does not guarantee average coverage or on-target specificity for each sample. We can instead recommend target data output that would increase the chances of obtaining the desired coverage based on the sample and library type utilized.

Data analysis is performed to the best of our ability and results are not guaranteed for samples or libraries not passing QC or do not fit the analysis pipeline’s criteria.

Yes, we can recommend the best data delivery option based on the platform and project details. Options include:

• Secure File Transfer Protocol (sFTP) - additional charges may apply
• Customer Cloud Account - AWS, Microsoft Azure, Google Cloud
• Hard Drive - additional charges may apply

Delivery to most customer cloud solutions is free of charge. If you have any questions, please submit an inquiry.

By default, results are sent via a secure File Transfer Protocol (sFTP). Please refer to our sFTP Data Download Guide for instructions on how to download your data and troubleshooting tips. Additional charges may be applicable for large data transfers.

Please refer to the delivery email sent from GENEWIZ for detailed information on the transfer and consult with your IT department to ensure compliance with your institution’s policies.

We take data security very seriously and we make every attempt to keep your data private and protected. The data transfer we offer is secure; however, should you desire an alternative delivery method, we are happy to work with you.

We hold any remaining samples for up to 3 months after project completion. Clinical samples processed in our Regulatory-Environment or CLIA-licensed laboratory may have longer sample storage timelines. Please contact us if you would like samples retained for a longer period or shipped back to you.

GENEWIZ offers raw data storage (i.e. FASTQ for Illumina, BAM for PacBio, POD5 for Oxford Nanopore) for up to 6 months after project completion. Data generated for samples processed in the Regulatory-Environment or CLIA-licensed laboratory may have longer data retention timelines. Please contact us if you would like the data to be retained for a longer period.

View our Sample Submission Guidelines for instructions on preparing and sending samples. Organize your samples in tubes or plates following the order indicated on the sample submission form.

• Tubes: Prepare samples in clearly labeled and well-organized 1.5 mL flip-cap microcentrifuge tubes. Please avoid using Parafilm to seal the tubes.

Unless otherwise instructed, GENEWIZ reserves the right to combine multiple vials of the same sample for extraction and/or library preparation.

• Plates: For projects with 16 or more samples, prepare samples in clearly labeled, securely sealed 96-well full-skirted PCR v-bottom plates. Arrange the samples vertically by column (i.e. A1, B1, C1, etc.)

Ship samples directly to our facility. Use the shipping address listed on the order receipt.

At GENEWIZ, we know that samples are valuable and not always perfect. We're here to help find solutions for working with less-than-ideal samples. Please reach out to us by submitting an inquiry, and we’ll work with you to meet your project needs.